Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

www.nature.com/scientificreports/ qualitatively and quantitatively discernible. The composition of the components may also be defined as a percentage, and they can be represented in color. MAPPING analysis (Fig. 2D, E, and F) shows that the conjugated component (OMV and DT) elements are also seen in the conjugate confirming the conjugation and formation of a new molecule.
The elemental composition maps based on EDAX analysis confirmed the existence of distinct elements in PA-OMVs-DT conjugate and conjugate components individually, as shown in (Fig. 2G and H). The findings revealed a minor difference in the percentages of conjugate and conjugate parts (numerically).

PA-OMVs-DT conjugate vaccination and survival rate of burnt mice infected with PAO1.
Schematic presentation of mice immunization is illustrated in Fig. 3A. The mice in the PBS group all died within the first three days of infection. All the unchallenged burnt mice in the control group survived. Groups vaccinated with the PA-OMVs-DT + adj conjugated exhibited higher survival rates (100%) than all other groups, significantly protecting immunized mice against PAO1 infection. Furthermore, immunization with PA-OMVs-DT without adjuvant and PA-OMVs + adj yielded a protective efficacy of 80 and 75%, respectively, higher than the PBS group. As shown in (Fig. 3B), PA-OMVs without adjuvant immunized mice have a partial protective role against PAO1 infection (60%) compared to the control group. No survival was noted in the toxoid-adj group (Fig. 3B).
Conjugated PA-OMVs vaccination protects mice with decreased bacterial burden. The bacterial loads in the liver, spleen, and blood of the mice groups vaccinated with conjugated and non-conjugated PA-OMVs, were lower than those in the PBS-administered group (Fig. 4A, B, and C, respectively). The PA-OMVs-DT + adj group had a considerably lower bacterial burden in the liver compared to the other groups, (Fig. 4A) (P < 0.0001). Similar findings were seen in spleen samples (Fig. 4B) (P < 0.0001). Furthermore, animals immunized with PA-OMVs-DT + adj had a lower bacterial burden in the spleen than mice immunized with PA-OMVs-DT without adjuvant (P < 0.05). This was not seen in the liver. The bacterial load in the skin was also reduced in the PA-OMVs-DT + adj vaccinated mice, although the differences were not significant (Fig. 4D). that the skin structure was disrupted, and the lack of nuclei in the injured tissues confirm a third-degree burn in mice with an average of 94.6 inflammatory cells/mm tissue in quantitative analysis by ImageJ software (Fig. 5A and B). The skin of the (PA-OMVs-DT + adj) vaccinated group exhibited eightfold less inflammatory cell infiltration of 12.6 cells/mm and less tissue damage following infection compared to the PBS group (Fig. 5C).
Immunization with PA-OMVs-DT + adj conjugate. The ELISA results revealed that there was no change in IgG levels before the first vaccination. Two weeks after the last vaccination (day 42), high IgG production was detected in all the immunized mice compared with control mice receiving PBS (Fig. 6A). The PA-OMVs-DT + adj conjugate group produced an IgG antibody level higher than the others (Fig. 6A). The addition of alum to the treatment increased IgG antibody production (P < 0.0001) in mice injected with PA-OMVs-DT + adj vs PA-OMVs-DT conjugate without adjuvant. Moreover, in the mice immunized with PBS as a control group, no antigen-specific IgG antibody was detected throughout the immunization process.
The concentrations of specific IgG1, IgG2a, and IgG2b antibodies as markers for Th2 and Th1 responses, were measured. Following the immunization of mice, the concentrations of specific IgG1, IgG2a, and IgG2b antibodies increased in all immunized mice (Fig. 6B, C, and D, respectively). Meanwhile, mice injected with PA-OMVs-DT + adj and PA-OMVs-DT conjugate showed significantly higher IgG1, IgG2a, and IgG2b production compared to other groups (P < 0.0001). Mice injected with PA-OMVs-DT + adj conjugate had significantly higher IgG1/IgG2a ratios than non-conjugated groups, with an IgG1/IgG2a ratio of about 1.5.

Discussion
OMVs are a potent vaccine candidates for inducing antibacterial protection 32 . DT was shown to increase the efficacy and protective effect of the vaccine in conjugation with alginate and lipopolysaccharide antigens of P. aeruginosa 33 . These findings are consistent with our findings that conjugating a carrier protein (DT) to PA-OMVs can improve the efficacy of OMV as a vaccine candidate.
However, previous studies indicated that OMVs can be used as a carrier protein and in human vaccines in terms of their license 34 . The present study showed better protectivity by PA-OMVs-DT conjugate than the other groups.
Significant components of OMVs from P. aeruginosa include OMPs, such as OprF, OprH, and OprG 35 . The protectivity of OMP as a vaccine candidate is well documented 21,36 . Meanwhile, flagellar proteins, composed of type b and c flagellins, have long been considered an ideal vaccine against P. aeruginosa infections 37 . Our data indicate that subcutaneouse vaccination of mice with both conjugated and non-conjugated PA-OMVs stimulated the humoral immune system compared to the PBS-administered group. Furthermore, the highest antibody level was observed following third dose. This is in concurrence with a previous study 37 . It has been shown that intramuscular administration of OMVs led to protection against models of A. baumannii sepsis and pneumonia 38 . It has been proposed that protection is via bacterial opsonisation 39 , and opsonizing antibodies were induced by intramuscular OMV delivery 38 . This is consistent with the findings of the present study. The  , blood (C), and skin (D) homogenized samples at 24 h, and concentrations were presented in CFU per biopsy sample or gram tissue (CFU/g). Data were presented as box plots, with the median and interquartile ranges indicated. One-way ANOVA was used to compare values. The difference between immunized and control mice groups was indicated as a p value. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant. www.nature.com/scientificreports/ evaluation of antibody isotype in mice groups showed that IgG1 was produced more than IgG2a and IgG2b in all immunized mice groups. Since the IgG1 subtype is predominant in the sera of immunized mice, as well as the pattern of IgG1/IgG2a responses, it is possible to explain that humoral immunity is the dominant immune response against P. aeruginosa in the burn infection model. Zhang et al. showed that mixed humoral and cellular immune responses were induced, followed by PAO1-derived OMVs immunization as a candidate vaccine 40 . They revealed that humoral immunity might be pivotal for PA-OMVs mediated protection. Lower bacterial loads in the blood, liver, and spleen of infected immunized mice suggest crucial role of immunization in reduction of the local and systemic dissemination of PAO1 from the infection site. This is further confirmed by histopathology results. Histopathology of skin tissue from the PBS-administered group revealed severe inflammation due to PAO1 subcutaneous injection, whereas immunization with conjugated-PA-OMV-DT + adj reduced inflammatory cell infiltration by eightfold, bleeding, and tissue damage. This may be in terms of the function and presence of the opsonic killing activity of antibodies against PAO1 in the burned wound site, which caused local immunity by decreasing the dissemination of PAO1. Enhancement of humoral immunity by local disruption or elimination of P. aeruginosa burden appears to be a priority in overcoming burn infection.
Although induction of IgG antibody titer is a good index of enhanced immunity in defense against bacterial infection, the efficiency and quality of these antibodies are more crucial in the opsonization and phagocytic death www.nature.com/scientificreports/ of this bacterium. Therefore, to determine the function of an opsonophagocytic of antibodies opsonophagocytosis assay was designed. The findings revealed that specific antibodies promoted opsonophagocytosis and killed PAO1 strain. High titer of opsonic antibodies was induced in mice immunized with non-conjugated and conjugated immunogens. This study showed that the immunization could produce opsonic antibodies that clear PAO1 in a dose-dependent manner, while by increasing the dose, the phagocytosis improves. One of the most crucial factors to evaluate the efficacy of vaccines is protection against infection challenges. In the mice burn model, enhancement in opsonophagocytosis of the opsonic antibody finally elevates the survival rate of PAO1-infected mice. Our data revealed that vaccination with conjugated and non-conjugated PA-OMVs can protect burned mice against PAO1 infection. Immunization with PA-OMV-DT + adj brought about 100% protection in mice challenged with PAO1. Immunizations with non-conjugated PA-OMVs + adj caused partial Data were presented as mean ± S.D. The difference among groups was compared using the two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant, respectively. www.nature.com/scientificreports/ protection against infections with PAO1 strain, and the survival rate of mice increased by about 75% compared to the control group. Overall, it seems that OMV-based vaccines could protect burned mice against PAO1 infection. While the development of OMV-based vaccines looks promising and is currently in its infancy, several challenges remain and need to be considered in further studies, such as the proteomics-based characterization of OMVs, increased yields of OMVs, and decreased LPS toxicity. Our findings indicated the use of conjugated OMVs-DT as a vaccine candidate increased the immunogenicity and protective efficacy against PAO1 in burn wound infection mice. In conclusion, our study provided the basis for subsequent studies into preventing P. aeruginosa infection in burn wounds by immunization and paved the way for further investigation. Mass production of OMVs, and concerns associated with the LPS toxicity are the potential hurdles or limitations of this study to a viable clinical product, that need to be resolved in further studies. These findings support further development of OMVs as a vaccine platform against P. aeruginosa and warrant further exploration of intranasal delivery as a route of immunization. Vaccine delivery route is an important consideration for vaccine development 41 .Mucosal vaccinations elicit good local mucosal and systemic immune responses and would be easier to administer than injectable vaccines, resulting in a decreased risk of infection 42,43 . Mucosal immunization may also lead to qualitatively better immune responses, for example in humans intranasal immunization induced upper airway IgA responses 42 .

Materials and methods
Approval for animal experiments. All experimental protocols were approved by the Ethics Committee of Shahed University (Tehran, Iran) vide approval certificate reference IR.SHAHED.REC.1398.057. All methods were carried out following relevant guidelines and regulations of the National Institute of Health guide for the care and use of laboratory animals (NIH Publication No_ 8023, revised 1978). We confirm that this study is reported in accordance with ARRIVE guidelines. Female BALB/c mice aged six to eight weeks were purchased from Razi Vaccine and Serum Research Institute. Mice were matched for age and sex, maintained under the same settings (12:12 h light/dark cycle, 22-23 °C, and 40% humidity) throughout the trial, and kept in specific pathogen-free (SPF) environments. All mice were fed with a standard antibiotic-free diet and water ad libitum.
Purification and characterization of PA-OMVs. OMVs isolation and purification from P. aeruginosa were, according to Siadat et al. 28 . Briefly, P. aeruginosa strain PAO1 was obtained from Molecular Microbiology Research Center of Shahed University, Tehran-Iran. The bacterial culture in 1 L of LB broth was maintained at 37 °C, shaking at 200 rpm to an optical density of 1.2. PAO1 cells were centrifuged at 6000 rpm for 30 min at 4 °C. The pellet was stabilized in a volume 7.5 times its wet weight with a 1.0 M Tris buffer containing 10 mM EDTA (w/v). The suspension was again centrifuged at 20,000 g for 1 h at 4 °C. The suspension was supplemented with a volume of 1:20 of 0.1 M Tris buffer solution containing EDTA and 100 g/L sodium deoxycholate. After 10 min, the pellet was suspended in deoxycholate; then, it was separated by ultracentrifuge at 125,000 × g for 2 h at 4 °C. The PA-OMVs were filtered through 0.22-mm filter (Millipore, USA) and stored at − 70 °C for further use. The total protein concentration of PA-OMVs was measured using the Nanodrop and Bradford assay. The protein sample pattern was evaluated on SDS-PAGE. Ultimately, the LAL assay (Lonza, Walkersville, MD, USA) was used to determine the amount of endotoxin.

Conjugation of OMVs-DT.
PA-OMVs were covalently conjugated to DT as a carrier protein using the adipic acid dihydrazide (ADH) (Sigma, USA) as a spacer molecule and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) (Sigma, USA) as a linker. At first, 2 mg/ml of PA-OMVs was exposed to EDAC (final concentration of 0.1 M), and then reacted with ADH for 5 h at pH: 8. Thin-layer chromatography (TLC) was used to monitor the reaction's solution for 6 h. The conjugation of PA-OMVs and ADH was then purified for 24 h using three water changes and a 2 kDa cut-off dialysis bag against water at 4 °C. Then, 2 mg/ml of this solution reacted with DT containing 1 mg/ml protein in the presence of EDAC for 24 h. Then, PA-OMVs-DT conjugate was purified via Sephadex G-750 gel filtration chromatography (Sigma, USA). The fractions were collected and centrifuged as a PA-OMVs-DT conjugate after being measured at 280 nm (protein) in the obtained fractions. Finally, the conjugated molecules were lyophilized in glass vials with a volume of 1 ml, passed through a 0.45 mm Millipore filter (USA), and kept at − 20 °C until injection.
Immunization of mice groups. The mice were divided into six groups; group I was injected with sterile PBS. Mice within group II received PA-OMVs (50 µg/mL), group III received conjugation of OMVs-DT formulated with alum adjuvant (Sigma, USA) (PA-OMVs-DT + adj), group IV received the conjugation of PA-OMVs-DT, group V received PA-OMVs formulated with alum adjuvant (PA-OMVs + adj), group VI received DT formulated with alum adjuvant (DT + adj), on days 0, 14 and 28. All the mice groups were immunized subcutaneously (SC), injecting 100 µl in groups without alum adjuvant and 200 µl in groups with alum adjuvant. Blood was obtained from the orbital sinus on days 0, 14, 28, and 42, and serum was collected for further analysis. The Control group consisted of non-immunized, non-infected burn mice.

Development of burn infection.
Two weeks after the last immunization, on day 42, the immunized mice were burned and challenged 36 . In brief, to create a burn, the back portions of the mice in each group were shaved at least 24 h before burn wound induction. Then, mice were anesthetized with the anesthetic drug Ketamine (100 mg/ml) and Xylazine (20 mg/ml) mixture. A third-degree burn wound was created by a custom-made cylindrical probe (20 × 20 × 100 mm, 150 g) heated by a gas flame to the temperature of 104 °C and put on the shaved part of the animal for 8 s. Immediately after burning, 500 μl of 0.9% saline was injected intraperitoneally (i.p.) into the burned mice for fluid replacement. Acetaminophen (0.25 mg/ml) was used as post-burn analgesic. Subsequently, the mice were infected by subcutaneous injection of a lethal dose (3 × 10 2 CFU) PAO1 at the burn center.

Determination of bacterial burden and survival rate.
Twenty-four hours after infecting the burnt wounds with 3 × 10 2 CFU of PAO1, the immunized and control mice groups were sacrificed. To determine the local dissemination of PAO1 strain in burn wound infections, burned skin at the injection site (15 × 15 mm) was collected. Moreover, to detect the systemic dissemination of PAO1, the infected mice's liver, spleen, and blood were aseptically collected. After that, the tissues were weighed, and sterile PBS was used to homogenize them. Then, homogenous samples were serially diluted in sterile PBS before being plated on Nutrient Agar (NA) and incubated for 24 h at 37 °C. The number of colony-forming units (CFUs) from each plate was then measured as CFUs per gram of tissue (CFU/g). The survival rate of experimental mice in each group was monitored for a 10-day.
Histological analysis. The skin samples were collected and fixed in 10% formalin, then embedded in paraffin, stained with Hematoxylin and Eosin (H&E), and observed under a light microscope. ImageJ is a well-known and publicly available image processing tool (http:// rsbweb. nih. gov/ ij/) released with many plugins and macros useful to biomedical image processing 45 . Automatic mammalian cell counting with ImageJ was previously reported 46 . In this study, an automated counting method utilizing ImageJ 1.53t (https:// imagej. nih. gov/ ij/ downl oad. html) was used for quantitative analysis of inflammatory cell infiltration. Five field images of each histogram showing various concentrations of inflammatory cell infiltration were studied.

Statistical analysis.
All experiments were performed in triplicate for each sample, and the results are expressed as mean ± standard deviation (SD). SPSS 24.0 (SPSS, Inc., USA) and GraphPad Prism version 9.4.1.681 (GraphPad Software, Inc., USA) were used to conduct statistical analyses and comparisons. A two-way analysis of variance (ANOVA) was used to assess the ELISA assays. Tukey's multiple comparison test was used. The oneway analysis of variance (ANOVA) used to analyze bacterial loads and opsonic killing action. The survival data were analyzed by Kaplan-Meier survival curves and log-rank test. P values < 0.05 were considered statistically significant ("Supplemnatary information").

Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.